本研究は、2022年度JST基礎研究(日本移植学会)の支援を受けて行われました。

この実験計画書は、京都大学の動物実験委員会で承認されました。この研究では、市販の供給源から入手した、10週間齢以上、体重25gから30gのC57BL/6マウスを利用しました( 「材料の表」を参照)。すべての動物に2.5%イソフルランを麻酔し(施設で承認されたプロトコルに従って)、特定の病原体を含まない条件下で維持し、すべての実験手順は京都大学の動物実験規則に準拠して実施されました。研究に使用された適切な機器は、 材料表 にリストされ、 図1 と 図2に示されています。
1.動物の選択
<ol><li>体重25〜30 gのマウスを使用してください。 注: マウス肝臓同種移植片2 は、主要な組織適合遺伝子複合体障壁を越えて一般に受け入れられていますが、この研究では、寒冷虚血再灌流障害10 の詳細なメカニズムに焦点を当てるために、同系 MOLT モデルを選択しました。体重が25g未満のマウスは、薄い胆管にインナーステントを挿入できないため、推奨されません。同様に、体重が30 gを超えるマウスは、血管の周囲に大量の腹腔内脂肪が存在するため、推奨されません。</li>
</ol>2.袖口作り
<ol><li>PVとIHIVC用にそれぞれ20Gと16Gの静脈カテーテルを準備します。</li>
	<li>メスを使ってカテーテルを切ってカフを作ります。袖口の本体の長さは2 mm、延長ハンドルは1 mmである必要があります(図3A)。 注意: ハンドルが大きすぎると、袖口の挿入が困難になる場合があります。</li>
	<li>袖口の表面に、メスの刃の裏側を使用して糸を固定するための浅い溝を作成します。 注意: メスの刃の裏側を袖口に当てるときは、刃の先端が約60度の角度になっていることを確認してください( 図3Bを参照)。最初の溝を作ったら、メスの刃を固定し、袖口を回転させます。理想的には2つの溝が推奨されますが、1つの溝でも問題なく十分です。</li>
</ol>3.袖口アタッチメント
<ol><li>プラスチック製の長方形の容器に、小さな角氷を置き、その上に小さな金属製のカップ(直径6cm)を置きます。カップに臓器保存液( 材料の表を参照)を入れ、その中に同系移植片を置きます。</li>
	<li>綿棒を使用して同系肝移植片(別のドナーマウスから抽出)を穏やかに転がし、肝門が上を向くようにします。IVCの内腔に溜まった血液を臓器保存液で完全に除去します。</li>
	<li>たとえば、脾臓静脈に取り付けられた糸を使用して、PVを袖口に通します( 図4Aを参照)。</li>
	<li>カフハンドルを12時の位置にして、ハンドルとカフの両方をブルドッグclで固定しますamp ( 材料の表を参照)、PVの端から2〜3mmの位置に配置します。</li>
	<li>大きな血管鉗子と固定型を使用してブルドッグクランプをさらに固定し、術野を確立します( 図4Bを参照)。</li>
	<li>PVルーメンを約15〜20倍の倍率で注意深く検査します。必要に応じて、水を滴下すると、内腔の視認性を高めることができます。</li>
	<li>PVルーメンをそっとつかみ、カフを通して外在化します。</li>
	<li>完全に外部化されたら、8-0 を使用して二重結紮で固定します。袖口の溝に沿った絹糸(図4C、D)。 注:結紮糸が緩んでいないことを確認してください。また、結紮点が大きすぎるとカフ挿入の邪魔になることもあるので注意が必要です。結紮点は任意の方向に配置できます。</li>
	<li>PVに使用したのと同様の手順を使用して、カフをIHIVCに取り付けます(図4E)。 注意: IHIVCをカフで固定するときは、肝実質に噛まないようにしてください。PVよりネックが短いので、クランプ位置を慎重に決めてください。</li>
	<li>IHIVCの基部に糸を通し、IHIVCを一時的に合字させることで、スリップノットを作成します(図5)。この糸は再灌流後に除去されます。</li>
</ol>4. 門脈再建
<ol><li>イソフルランを2.5%で麻酔を誘発し、それを0.6%に減らしてから、公開された文献2に記載されているように、天然の肝臓を抽出します。開腹術の前に、ヨードフォアと70%エタノールを交互に繰り返して手術部位を少なくとも3回消毒します。</li>
	<li>肝葉が適切に配置され、カフ付きPVにねじれやねじれがないことを確認してください。</li>
	<li>ピーン鉗子を使用して受信者のPVエッジをそっとつかみ、万力で固定します。 注:この記事では、万力とは、ペアン鉗子をテーブルに固定するために使用される機械装置を指します。万力の詳細については、 材料の表を参照してください。</li>
	<li>レシピエントのPVに血管クランプを適用し、8-0を渡しますその周りの絹糸。</li>
	<li>PVエッジから約1mmの円周セクションを0.5mm作成します(図6A)。専用の器具(先端を切り取り、L字型に曲げた27Gの針)を使用して、生理食塩水を内腔に通しながら穴を拡大します。</li>
	<li>まっすぐな鉗子を使用してカフハンドルをつかみ、内腔に挿入します。</li>
	<li>完全に挿入したら、カフを8-0で固定します。絹糸。固定には1回の結紮で十分です。</li>
	<li>血管クランプを取り外し、肝臓を再灌流します。.</li>
	<li>ピーン鉗子を慎重に離し、門脈再建を完了します。PVの再構築プロセス全体には約5分かかります。</li>
</ol>5. 下大静脈下再建
<ol><li>肝葉を適切に配置し、レシピエントIHIVC背側に取り付けられたピーン鉗子を移植片の右下葉に動かし、万力で固定します。</li>
	<li>レシピエントのIVIHCの周囲に残っている肝臓組織を、綿棒で優しくこすって取り除きます。</li>
	<li>8-0で合格受信者のIVIHCの周りの絹糸。</li>
	<li>レシピエントのIVIHCエッジから約1mmに4/0.5円周セクションを作成します(図6B)。</li>
	<li>通常の生理食塩水を内腔に導入した後、カフを内腔に挿入します。 注:首が短いため、袖口はPVよりも浅く挿入されることがよくあります。</li>
	<li>8-0で袖口を慎重に固定します絹糸を通し、血管クランプとピーン鉗子を取り外します。</li>
</ol>6.術後ケア
<ol><li>加熱パッドと加温ランプを利用して、術後の回復を促進します。</li>
	<li>血圧、心拍数、呼吸数、体温、および食物と水の消費量を継続的に監視します。</li>
	<li>術後の痛みを軽減するためのカルプロフェンの皮下注射(5 mg / kg、6時間ごとまたは必要になるまで)を投与します。地域の機関のガイドラインに従ってください。 注:マウスの状態が悪化した場合、実験は直ちに中止され、マウスは二酸化炭素の吸入によって安楽死させられました。</li>
	<li>組織サンプルを採取するには、マウスを再麻酔し、IACUC承認の方法で安楽死させます。</li>
</ol>

マウス肝移植における効率的な血管吻合のための戦略

<ol>
	<li>Qian, S. G., Fung, J. J., Demetris, A. V., Ildstad, S. T., Starzl, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+liver+transplantation+in+the+mouse.">Orthotopic liver transplantation in the mouse.</a> Transplantation. 52 (3), 562-564 (1991).</li><li>Yokota, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+mouse+liver+transplantation+to+study+liver+biology+and+allograft+tolerance.">Orthotopic mouse liver transplantation to study liver biology and allograft tolerance.</a> Nat Protoc. 11 (7), 1163-1174 (2016).</li><li>Shen, X. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+responses+in+a+new+mouse+model+of+prolonged+hepatic+cold+ischemia+followed+by+arterialized+orthotopic+liver+transplantation.">Inflammatory responses in a new mouse model of prolonged hepatic cold ischemia followed by arterialized orthotopic liver transplantation.</a> Liver Transpl. 11 (10), 1273-1281 (2005).</li><li>Kageyama, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemia-reperfusion+injury+in+allogeneic+liver+transplantation:+A+role+of+CD4+T+cells+in+early+allograft+injury.">Ischemia-reperfusion injury in allogeneic liver transplantation: A role of CD4 T cells in early allograft injury.</a> Transplantation. 105 (9), 1989-1997 (2021).</li><li>Li, T., Hu, Z., Wang, L., Lv, G. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Details+determining+the+success+in+establishing+a+mouse+orthotopic+liver+transplantation+model.">Details determining the success in establishing a mouse orthotopic liver transplantation model.</a> World J Gastroenterol. 26 (27), 3889-3898 (2020).</li><li>Tian, Y., R&#252;diger, H. A., Jochum, W., Clavien, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+arterialized+and+nonarterialized+orthotopic+liver+transplantation+in+mice:+Prowess+or+relevant+model.">Comparison of arterialized and nonarterialized orthotopic liver transplantation in mice: Prowess or relevant model.</a> Transplantation. 74 (9), 1242-1246 (2002).</li><li>Steger, U., Sawitzki, B., Gassel, A. M., Gassel, H. J., Wood, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+hepatic+rearterialization+on+reperfusion+injury+and+outcome+after+mouse+liver+transplantation.">Impact of hepatic rearterialization on reperfusion injury and outcome after mouse liver transplantation.</a> Transplantation. 76 (2), 327-332 (2003).</li><li>Chen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+various+techniques+of+mouse+liver+transplantation.">A review of various techniques of mouse liver transplantation.</a> Transplant Proc. 45 (6), 2517-2521 (2013).</li><li>Kojima, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+carcinoembryonic+antigen-related+cell+adhesion+molecule+1-T+cell+immunoglobulin+domain+and+mucin+domain-containing+protein+3+cross+talk+alleviates+liver+transplant+injury+in+mice+and+humans.">T cell carcinoembryonic antigen-related cell adhesion molecule 1-T cell immunoglobulin domain and mucin domain-containing protein 3 cross talk alleviates liver transplant injury in mice and humans.</a> Gastroenterology. 165 (5), 1233-1248 (2023).</li><li>Yokota, S., Yoshida, O., Ono, Y., Geller, D. A., Thomson, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+transplantation+in+the+mouse:+Insights+into+liver+immunobiology,+tissue+injury,+and+allograft+tolerance.">Liver transplantation in the mouse: Insights into liver immunobiology, tissue injury, and allograft tolerance.</a> Liver Transpl. 22 (4), 536-546 (2016).</li></ol>

開示すべき利益相反のある著者はいません。

血管再建を学ぶことは、MOLTを成功させるための最も困難な側面です。カフの品質は、マウスのサイズが小さいことを考えると、再建の難しさに大きく影響します5。この記事では、カフの準備、取り付け、および再建の詳細なプロトコルを提供します。
カフの準備と接続に関する以前のレポートと大きな違いはありませんが、いくつか...

マウス同所性肝移植(MOLT)は、1991年に最初に報告された効果的な実験方法です1。遺伝子改変マウスや各種研究用試薬を用いたこの実験モデルは、温冷虚血や再灌流障害の研究に重要な役割を果たしてきました。しかし、このモデルの高い技術的複雑さは、肝移植のための基礎医学の開発を妨げてきました2。MOLTは、(1)ドナーマウスからの肝臓の回収、(2)バックテーブル手術、(3)レシピエントへの肝臓移植の3つの主要なステップから成ります。これらのレシピエント手順の中で、血管吻合は最大の課題をもたらします。下大静脈上吻合は通常、手縫い2で完了しますが、下肝下大静脈(IHIVC)と門脈(PV)は、手縫いの縫合の代わりにプラスチック製の袖口を使用してより効率的に再建できます。
無肝期は、レシピエントの天然肝臓の除去と移植片の移植片の移植の間の間隔を意味します。一貫した結果を得るには、無肝時間を20分未満に制限することが不可欠です。その結果、このモデルを採用した研究は、特定の機関に限定されている3,4,5,6,7,8,9。MOLTの様々な段階の中で、PVとIHIVCのスムーズな再建を実現することは、無肝時間を最小化し、移植を成功させるために重要です。
PVおよびIVC再建は、手縫い縫合糸2,5,8と比較して、カフ技術が血管吻合を簡素化するため、一般に血管カフを使用して行われます。血管カフの準備とカフの確実な取り付けに関連する技術は、レシピエントの血管再建の複雑さに大きな影響を与えます。私たちの目的は、カフ法に関連する多くの技術的なヒントを詳細に視覚的に指導し、それによって学習曲線を短縮することです。これらのビデオ クリップは、カフを血管に取り付け、レシピエント手術中に PV と IHIVC を再構築する方法を明確に理解します。

<table><tbody><tr><td>16 G intravenous catheter</td><td>TERUMO</td><td>SR-FF2032</td><td>IHIVC cuff</td></tr><tr><td>20 G intravenous catheter</td><td>TERUMO</td><td>SR-FF1651</td><td>PV cuff</td></tr><tr><td>8-0 braid silk</td><td>Natsume Seisakusho</td><td>CR9-80B2</td><td>8-0 silk</td></tr><tr><td>Belzer UW Cold Storage Solution&amp;nbsp;</td><td>Astellas</td><td></td><td>Organ preservation fluid</td></tr><tr><td>Bulldog clamp</td><td>B BRAUN</td><td>FB329R</td><td>Bulldog clamp</td></tr><tr><td>C57BL/6 mice&amp;nbsp;&amp;nbsp;</td><td>Oriental Bio Service</td><td></td><td></td></tr><tr><td>Isoflurane inhalation solution</td><td>Viatris</td><td></td><td>Anesthesic</td></tr><tr><td>Micro Blunted Tips 0.1 mm x 0.06 mm&amp;nbsp;</td><td>F.S.T</td><td>11253-20</td><td>Straight microforceps</td></tr><tr><td>Micro Serrefine Clamp Applicator with Lock</td><td>F.S.T</td><td>18056-14</td><td>Vessel clip applicator</td></tr><tr><td>Micro Serrefines&amp;nbsp;</td><td>F.S.T</td><td>18055-4</td><td>Vessel clip</td></tr><tr><td>No.11 Spare Blades</td><td>FEATHER Safety Razor</td><td>11</td><td>Blades</td></tr><tr><td>Ophthalmic scissor, round handle</td><td>B BRAUN</td><td>FD103R</td><td>Microscissor</td></tr><tr><td>Plastic rectangular-shaped container&amp;nbsp;&amp;nbsp;</td><td>Daiso</td><td></td><td>10 cm long, 15 cm wide and 6 cm high</td></tr><tr><td>SuperGrip Tips</td><td>F.S.T</td><td>00649-11&amp;nbsp;</td><td>Curved microforceps</td></tr><tr><td>SZX7</td><td>Olympus</td><td>SZX7</td><td>Microscope</td></tr><tr><td>Vise</td><td>Niigataseiki</td><td>00505351</td><td>A mechanical tool to secure Pean forceps on the table</td></tr></tbody></table>

vascular reconstruction with the cuff technique in mouse orthotopic liver transplantation

マウス同所性肝移植は、肝虚血や再灌流障害の根底にあるメカニズムを調べるための有効な方法論です。しかし、技術的な課題は、この貴重な実験モデルを活用し、これらのスキルを次世代に継承する上での障壁となっています。この手順の最も困難な側面は、門脈 (PV)、下肝下大静脈 (IHIVC)、および下大静脈上を含む血管再建です。縫合糸ではなくプラスチック製のカフを使用することで、PVやIHIVCの再建をよりスムーズに行うことができます。静脈カテーテルで作ったカフを移植血管の先端に取り付け、カフをレシピエント血管に介在させることで血管を再構築します。最も重要な2つの側面は、血管の内腔を適切に視覚化することと、過度の力の使用を避けることです。私たちの目的は、レシピエント手術におけるカフ技術を使用した血管再建の技術的概要を提供することです。カフ技術に関するこれらの技術的ヒントは、顕微外科医が血管再建を容易にし、研究を前進させるのに役立つと期待されています。

PV再建は、門脈のクランプを外したときに曲がりくねった状態がなく、肝臓が均一に灌流されている場合に成功します。無肝時間が25分を超えるとマウスの死亡リスクが高まるため、無肝時間は20分未満にする必要があります。IHIVCの再建は、移植片からの血液逆流がない場合、成功したと見なされます。
臓器保存溶液を使用して移植片を低温で1時間保存すると、再灌流?...

Watch this Scientific Journal Video about Author Spotlight: Exploring Cold Ischemia and Warm Reperfusion Injury in Fatty Liver Transplantation at JoVE.com

著者スポットライト:脂肪肝移植における冷虚血と温再灌流障害の探索

このプロトコルは、マウス同所性肝移植におけるカフ技術を使用した血管再建のための技術情報を提供します。

マウス同所性肝移植におけるカフ技術による血管再建

Mouse orthotopic liver transplantation is an effective methodology for investigating the underlying mechanisms of liver ischemia and reperfusion injury. ...

肝移植、特に低温虚血と温再灌流障害の研究に重点を置いています。そのための研究ツールとして、マウス肝移植モデルを用いています。テクニックが難しいため、習得して一貫した結果を達成するには長い時間がかかる場合があります。将来的には、脂肪肝移植の研究も行っていきたいと考えています。これは、臨床肝移植で克服すべき問題です。

vascular-reconstruction-with-cuff-technique-mouse-orthotopic-liver

Research

JoVE Journal

Medicine

Vascular Reconstruction with the Cuff Technique in Mouse Orthotopic Liver Transplantation

1.2K ビュー.  Kyoto University. 肝移植、特に低温虚血と温再灌流障害の研究に重点を置いています。そのための研究ツールとして、マウス肝移植モデルを用いています。テクニックが難しいため、習得して一貫した結果を達成するには長い時間がかかる場合があります。将来的には、脂肪肝移植の研究も行っていきたいと考えています。これは、臨床肝移植で克服すべき問題です。

ビデオ: 著者スポットライト:脂肪肝移植における冷虚血と温再灌流障害の探索

Orthotopic Liver Transplantation in Rats

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat liver transplantation in 1979 by Kamada et al.3, this model has remained the gold standard despite various proposed alternative techniques4. Nevertheless, its broader use is limited by its steep learning curve5.
In this video paper, we show a simple and easy-to-establish revision of Kamada's two-cuff technique. The suprahepatic vena cava anastomosis is performed manually with a running suture, and the vena porta and infrahepatic vena cava anastomoses are performed utilizing a quick-linker cuff system6. Manufacturing the quick-linker kit is shown in a separate video paper.

We present an easy-to-establish revision of the classical two-cuff technique for orthotopic liver transplantation in rat.

ラット同所性肝移植

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat ...

医学

Murine Cervical Heart Transplantation Model Using a Modified Cuff Technique

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are available to perform mechanistic in vivo studies. While heart transplantation models using a suture technique were first successfully developed in rats, the translation into an equally widespread used murine equivalent was never achieved due the technical complexity of the microsurgical procedure. In contrast, non-suture cuff techniques, also developed initially in rats, were successfully adapted for use in mice1-3. This technique for revascularization involves two major steps I) everting the recipient vessel over a polyethylene cuff; II) pulling the donor vessel over the formerly everted recipient vessel and holding it in place with a circumferential tie. This ensures a continuity of the endothelial layer, short operating time and very high patency rates4.
Using this technique for vascular anastomosis we performed more than 1,000 cervical heart transplants with an overall success rate of 95%. For arterial inflow the common carotid artery and the proximal aortic arch were anastomosed resulting in a retrograde perfusion of the transplanted heart. For venous drainage the pulmonary artery of the graft was anastomosed with the external jugular vein of the recipient5.
Herein, we provide additional details of this technique to supplement the video.

The murine cervical heart transplantation model is well suited for immunological as well as ischemia reperfusion injury studies. We modified the procedure using a non-suture cuff technique and performed more than 1,000 successful transplants with this approach.
Herein, we provide additional details of this technique to supplement the video.

変更されたカフ法を用いたマウス子宮頸心臓移植モデル

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are ...

Heterotopic Cervical Heart Transplantation in Mice

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies this model by avoiding challenging suture anastomoses of small vessels thereby reducing warm ischemia time. In comparison to abdominal graft implantation the cervical model is less invasive and the implanted graft is easily accessible for further follow-up examinations. Anastomoses are performed by pulling the ascending aorta of the graft over the cuff with the recipient&#8217;s common carotid artery and by pulling the main pulmonary artery over the cuff with the external jugular vein. Selection of appropriate cuff size and complete mobilization of the vessels are important for successful revascularization. Ischemia-reperfusion (I/R) injury can be minimized by perfusing the graft with a cardioplegic solution and by hypothermia. In this article, we provide technical details for a simplified and improved cuff technique, which should allow surgeons with basic microsurgical skills to perform the procedure with a high success rate.

The cuff technique shortens and facilitates the model of heterotopic cervical heart transplantation in mice by avoiding technically challenging suture anastomoses of small vessels. The technical details shown in this video paper should allow researches to establish this model in their laboratories.

マウスにおける異子宮頸心臓移植

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies ...

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. To better understand the mechanism behind these surgical procedures, a rat partial liver transplantation (PLTx) model was established for relevant surgical studies. Because of the complexity of the rat PLTx model, a protocol with detailed descriptions is required. An article published previously reported a protocol in which ex vivo hepatectomy was used to achieve 50% rat PLTx. In contrast to this protocol, we introduced a re-arterialized PLTx with an in vivo 70% hepatectomy. An updated vessel-oriented hepatectomy was incorporated into the rat PLTx to refine the microsurgical procedure. The portal veins and hepatic arteries of the left lateral lobe and the median lobe were individually dissected and ligated before removal of the liver parenchyma, thereby decreasing the probability of bleeding in the remnant liver stump. Furthermore, an end-to-side vessel anastomosis between the common hepatic artery and the enlarged proper hepatic artery was introduced to re-arterialize the hepatic artery. By using this end-to-side vessel anastomosis technique, the diameter of the anastomosis was enlarged, thereby decreasing the difficulty of hand suture and maintaining a high rate of anastomotic patency. Moreover, the cuff anastomosis of the infrahepatic vena cava was slightly modified. A section of circumferential liver parenchyma around the vena cava of a recipient was preserved during cuff anastomosis to maintain the three-dimensional shape of the vascular lumen. This section of liver parenchyma was removed after completing the anastomosis. With this modification, the step involving placement of stay sutures was omitted, thereby further shortening the cuff anastomosis time. By using this protocol of rat PLTx, a low liver enzyme level, an intact liver lobular architecture and a high survival rate were achieved after microsurgery.

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Here, a protocol involving re-arterialized rat partial liver transplantation is presented. Specifically, 70% liver was resected in vivo by using an updated technique of vessel-oriented hepatectomy. The hepatic artery was reconstructed in an end-to-side manner. The cuff technique was modified to shorten the anastomosis time of the infrahepatic vena cava.

再門脈部分肝移植のラット体内血管指向 70% 肝切除

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. ...

A Rat Model of Orthotopic Liver Transplantation Using a Novel Magnetic Anastomosis Technique for Suprahepatic Vena Cava Reconstruction

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep learning curve. The introduction of the cuff technique for anastomosis of the portal vein (PV) and infrahepatic vena cava (IHVC) has significantly simplified the transplant procedure in rats. However, due to the short anterior wall of the recipients' suprahepatic vena cava (SHVC), the cuff technique is very difficult to use for the reconstruction of the SHVC. Most researchers in this field still use the hand-suture technique for SHVC reconstruction, which makes it the bottleneck step in rat orthotopic liver transplantation. The magnetic anastomosis technique (i.e., magnamosis) is a method of connecting two vessels using the attractive force between two magnets. Our recent study has shown that the magnetic anastomosis technique is superior to the hand-suture technique for SHVC reconstruction in rats. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using the novel magnetic anastomosis technique. In this model, the reconstruction of the PV and IHVC was performed by the standard cuff technique, while the reconstruction of the bile duct (BD) was performed by a stent technique. The hepatic re-arterialization was not performed. The magnetic anastomosis technique made SHVC reconstruction much easier and significantly shortened the anphepatic phase. After a reasonable learning curve, even researchers without advanced microsurgical skills can produce reliable and reproducible results using this rat model of OLT.

The reconstruction of the suprahepatic vena cava (SHVC) remains a difficult step in rat orthotopic liver transplantation. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using a novel magnetic anastomosis technique.

同所性同種のモデルラット Suprahepatic 上大静脈再建のため新規磁気吻合法を用いた肝移植

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep ...

Optimization of the Cuff Technique for Murine Heart Transplantation

Murine cardiac transplantation has been performed for more than 40 years. With advancements in microsurgery, certain new techniques have been used to improve surgical efficiency. In our lab, we have optimized the cuff technique with two major steps. First, we used the inner tube technique to insert a temporary inner tube into the external jugular vein and carotid artery blood vessel to facilitate eversion of the vessel over the cuff. Second, we performed complete heterotopic cardiac transplantation through the collaboration of two experienced surgeons. These modifications effectively reduced the operation time to 25 minutes, with a success rate of 95%. In this report, we describe these procedures in detail and provide a supplemental video. We believe that this report on the improved cuff technique will offer practical guidance for murine heterotopic heart transplantation and will enhance the utility of this mouse model for basic research.

We introduce an inner tube approach to the cuff technique for mouse cervical heterotopic heart transplantation to help evert the vessel over the cuff. We found that cooperation between two experienced surgeons markedly shortens the operation time.

マウスの心臓移植のためのカフ技術の最適化

Murine cardiac transplantation has been performed for more than 40 years. With advancements in microsurgery, certain new techniques have been used to ...

免疫・感染学

Mouse Heterotopic Cervical Cardiac Transplantation Utilizing Vascular Cuffs

Murine models of cardiac transplantation are frequently utilized to study ischemia-reperfusion injury, innate and adaptive immune responses after transplantation, and the impact of immunomodulatory therapies on graft rejection. Heterotopic cervical heart transplantation in mice was first described in 1991 using sutured anastomoses and subsequently modified to include cuff techniques. This modification allowed for improved success rates, and since then, there have been multiple reports that have proposed further technical improvements. However, translation into more widespread utilization remains limited due to the technical difficulty associated with graft anastomoses, which requires precision to achieve adequate length and caliber of the cuffs to avoid vascular anastomotic twisting or excessive tension, which can result in damage to the graft. The present protocol describes a modified technique for performing heterotopic cervical cardiac transplantation in mice which involves cuff placement on the recipient's common carotid artery and the donor's pulmonary artery in alignment with the direction of the blood flow.

Mouse cardiac transplantation models represent valuable research tools for studying transplantation immunology. The present protocol details mouse heterotopic cervical cardiac transplantation that involves the placement of cuffs on the recipient's common carotid artery and the donor's pulmonary artery trunk to allow for laminar blood flow.

血管カフを用いたマウスヘテロトピック頸部心臓移植

Murine models of cardiac transplantation are frequently utilized to study ischemia-reperfusion injury, innate and adaptive immune responses after ...

Murine Cervical Aortic Transplantation Model using a Modified Non-Suture Cuff Technique

With the introduction of powerful immunosuppressive protocols, distinct advances are possible in the prevention and therapy of acute rejection episodes. However, only minor improvement in the long-term results of transplanted solid organs could be observed over the past decades. In this context, chronic allograft vasculopathy (CAV) still represents the leading cause of late organ failure in cardiac, renal and pulmonary transplantation.
Thus far, the underlying pathogenesis of CAV development remains unclear, explaining why effective treatment strategies are presently missing and emphasizing a need for relevant experimental models in order to study the underlying pathophysiology leading to CAV formation. The following protocol describes a murine heterotopic cervical aortic transplantation model using a modified non-suture cuff technique. In this technique, a segment of the thoracic aorta is interpositioned in the right common carotid artery. With the use of the non-suture cuff technique, an easy to learn and reproducible model can be established, minimizing the possible heterogeneity of sutured vascular micro anastomoses.

Here, we present a protocol of heterotopic aortic transplantation in mice using the non-suture cuff technique in a cervical murine model. This model can be used to study the underlying pathology of chronic allograft vasculopathy (CAV) and can help evaluate new therapeutic agents in order to prevent its formation.

改変非縫合カフ技術を用いたマウス子宮頸動脈大動脈移植モデル

With the introduction of powerful immunosuppressive protocols, distinct advances are possible in the prevention and therapy of acute rejection episodes. ...

Reduced Complications after Arterial Reconnection in a Rat Model of Orthotopic Liver Transplantation

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation of human liver transplantation due to the absence of arterial reconnection. Described here is a modified transplantation procedure that includes the incorporation of hepatic artery (HA) reconnection, leading to a marked improvement in transplant outcomes. With a mean anhepatic time of 12 min and 14 s, HA reconnection results in improved perfusion of the transplanted liver and an increase in long-term recipient survival from 37.5% to 88.2%. This protocol includes the use of 3D-printed cuffs and holders to connect the portal vein and infrahepatic inferior vena cava. It can be implemented for studying multiple aspects of liver transplantation, from immune response and infection to technical aspects of the procedure. By incorporating a simple and practical method for arterial reconnection using a microvascular technique, this modified rat OLT protocol closely mimics aspects of human liver transplantation and will serve as a valuable and clinically relevant research model.

The goal of this study is to modify the rat orthotopic liver transplant model to better represent human liver transplantation and improve recipient survival. The presented method reestablishes hepatic arterial inflow by connecting the donor liver&#39;s common hepatic artery to the recipient liver&#39;s proper hepatic artery.

ラット型異形肝移植モデルにおける動脈リコネクション後の合併症の減少

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation ...

A Modified Cuff Technique for Mouse Cervical Heterotopic Heart Transplantation Model

Cardiac allograft rejection limits the long-term survival of patients after heart transplantation. A mouse heart transplantation model is ideal for investigating the mechanism of cardiac allograft rejection in preclinical studies because of their high homology with human genes. This understanding would help develop unique approaches to improving patients&#39; long-term survival treated with cardiac allografts. In a mouse model, abdominal donor heart implantation is commonly performed with an end-to-end&#160;anastomosis to the recipient&#39;s aorta and inferior vena cava using stitches. In this model, the donor&#39;s heart is implanted by end-to-end anastomosis to the recipient&#39;s carotid artery and jugular vein by the modified-Cuff technique. The transplantation surgery is performed without stitching and thus may increase the survival of the recipient since there is no interference with the blood supply and venous reflux of the lower body. This mouse model would help investigate the mechanisms underlying the immunological and pathological (acute/chronic) rejection of cardiac allografts.

In the present protocol, a mouse heart transplantation model is used for investigating the mechanism of cardiac allograft rejection. In this heterotopic heart transplantation model, operation efficiency is improved, and the survival of cardiac grafts is ensured by a cervical end-to-end anastomosis of heart implantation using a modified Cuff technique.

マウス頸部異所性心臓移植モデルのための修正カフ法

Cardiac allograft rejection limits the long-term survival of patients after heart transplantation. A mouse heart transplantation model is ideal for ...